The discovery that RNA can catalyze chemical reactions has led to the identification of many new biological and biochemical roles for RNAs in cells. However despite significant effort, there is presently little information on the three-dimensional structures of RNA. This situation is in contrast to proteins where X-ray or NMR structures of hundreds of biologically active enzymes are known and these data are routinely used to help unravel the molecular mechanisms of action of these molecules. In this proposal state-of-the-art heteronuclear multi-dimensional NMR spectroscopy will be used to probe the structure and dynamics of a variety of biologically important RNAs and RNA structural motifs. Part of this project entails development and optimization of heteronuclear 2D, 3D and 4D NMR techniques for resonance assignment and structure determinations of RNAs. Similar techniques have revolutionized the solution structure determinations of proteins but they have yet to be applied to nucleic acids. Recent advances in the methodology for generating milligram quantities of 13C and/or 15N labelled RNAs, will now make it possible to perform more detailed structure determination of larger RNAs. One goal of this project focuses on identification and structure determination of RNA folding motifs. The following systems will be studied: i) RNA hairpins belonging to the frequently occurring GNRA (where N is any nucleotide and R is A or G) and CUUG tetraloop families; ii) RNA triplexes; and iii) naturally occurring RNA duplexes that contain internal loops or 'mismatched' base pairs. The studies will give us a wealth of new information on the building blocks of RNA tertiary structure. To investigate importance of RNA structural motifs in biological functions a protein-RNA complex consisting of a fragment of the signal recognition particle (SRP) RNA containing and its site-specific binding protein, the SRP-19 protein, will be studied by NMR. This RNA binding site contains a GNRR tetraloop and this system will be used to determine how the binding of this 19kD protein affects the structure of the RNA hairpin. These result will lead to a better understanding of the role of RNA structural motifs in protein-RNA recognition.